Optimized Synthetic human insulin gene was preferred to easy of cloning, plasmid stability, and protein expression away from the native sequence and its rare codons. Two steps to obtain the insulin, so we assembled the gene of 293 bp using a battery of overlapped synthetic oligos, then cloned into pET101directional TOPO expression vector downstream to the T7 promoter. The proinsulin products were produced as inclusion bodies in E. coli at a level of 10%. The batch cultivation of the strain yielded 6 g/L, while the high cell density of fed-batch cultivation yielded 46 g/L. The proinsulin purification yielded 110 mg/gram cell weight, and 1.3 mg/gram of a bioactive insulin. The native insulin was generated by enzymatic conversion of chemically processed proinsulin. The produced insulin was matched with that of a commercial aqueous version at a level of enzyme immunoassys, SDS-PAGE, RP-HPLC, and bioactivity. The present results showed that the produced insulin has a comparable biochemical and potency similar to that of commercial one.
Of ten actinobacterial isolates, Streptomyces cellulosae Actino 48 exhibited the strongest suppression of Sclerotium rolfsii mycelium growth and the highest chitinase enzyme production (49.2 U L-1 min-1). The interaction between Actino 48 and S. rolfsii was studied by scanning electron microscope (SEM), which revealed many abnormalities, malformations, and injuries of the hypha, with large loss of S. rolfsii mycelia density and mass. Three talc-based formulations with culture broth, cell-free supernatant, and cell pellet suspension of chitinase-producing Actino 48 were characterized using SEM, Fourier transform infrared spectroscopy (FTIR), and a particle size analyzer. All formulations were evaluated as biocontrol agents for reducing damping-off, root rot, and pods rot diseases of peanut caused by S. rolfsii under greenhouse and open-field conditions. The talc-based culture broth formulation was the most effective soil treatment, which decreased the percentage of peanut diseases under greenhouse and open-field conditions during two successive seasons. The culture broth formulation showed the highest increase in the dry weight of peanut shoots, root systems, and yielded pods. The transcriptional levels of three defense-related genes (PR-1, PR-3, and POD) were elevated in the culture broth formulation treatment compared with other formulations. Subsequently, the bio-friendly talc-based culture broth formulation of chitinase-producing Actino 48 could potentially be used as a biocontrol agent for controlling peanut soil-borne diseases caused by S. rolfsii.
Viral plant diseases represent a serious problem in agricultural production, causing large shortages in the production of food crops. Eco-friendly approaches are used in controlling viral plant infections, such as biocontrol agents. In the current study, Streptomyces cellulosae isolate Actino 48 is tested as a biocontrol agent for the management of tobacco mosaic virus (TMV) and inducing tomato plant systemic resistance under greenhouse conditions. Foliar application of a cell pellet suspension of Actino 48 (2 × 107cfu. mL−1) is performed at 48 h before inoculation with TMV. Peroxidase activity, chitinase activity, protein content, and the total phenolic compounds are measured in tomato leaves at 21 dpi. On the other hand, the TMV accumulation level and the transcriptional changes of five tomato defense-related genes (PAL, PR-1, CHS, PR-3, and PR-2) are studied. Treatment with Actino 48 before TMV inoculation (48 h) induced tomato plants to increase their levels of peroxidase and chitinase enzymes. Furthermore, a significant increase in the concentration of total phenolic compounds was observed in Actino 48 and TMV-treated tomato plants compared to TMV-treated tomato plants alone. Treatment with Actino 48 reduced the TMV accumulation level (53.8%) compared to treatment with the virus alone. Actino 48 induced plant growth, where the fresh and dry weights of tomato plants increased. Additionally, significant increases of the PAL, PR-1, CHS, and PR-3 transcripts were observed. On the other hand, a higher induction of PR-2 was only observed in TMV-treated tomato plants. In conclusion, S. cellulosae isolate Actino 48 can be used as a biocontrol agent for the reduction of symptoms and severity of TMV.
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